Localized detection of glioma glycolysis using edited 1H MRS.

In vivo 1H MRS can be used to detect and quantify the lactate resonance at 1.3 ppm provided that overlapping lipid resonances are eliminated. A homonuclear spectral editing method was developed to acquire uncontaminated 1H spectra of lactate with adiabatic pulses. An advantage of the adiabatic pulse sequence is the ability to induce uniform flip angles and to maximize sensitivity in applications employing surface coil transmitters which produce highly inhomogeneous B1. Glycolytic activity in an intracerebral C6 glioma in rats was monitored by using adiabatic editing sequences to observe [3-13C]lactate produced from infused [1-13C]glucose. Acute hyperglycemia (serum glucose > 22 mM, n = 10) had no significant effect (P = 0.08) on the total ([12C] + [13C]) tumor lactate signal intensity.

Viability of Giardia cysts suspended in lake, river, and tap water.

Numerous waterborne outbreaks of giardiasis have occurred since 1965, yet little or no information has been reported on the viability of Giardia cysts in different aquatic environments. We have studied the viability of Giardia muris cysts suspended in lake, river, and tap water, while also monitoring water temperature, dissolved oxygen, pH, and other water quality parameters. Fecal pellets containing G. muris cysts were placed in glass vials covered with filter paper and exposed to (i) lake water at 15 ft (ca. 4.6 m) and 30 ft (ca. 9.2 m), (ii) river water, (iii) tap water, and (iv) distilled water stored under laboratory conditions. At 3, 7, 14, 28, 56, and 84 days, two vials from each environment were removed, and cyst viability was determined by (i) fluorogenic dye exclusion, (ii) production of giardiasis in an animal, and (iii) cyst morphology by Nomarski microscopy. In the fall, the cysts suspended at 30 ft in lake water remained viable for up to 56 days whereas cysts stored at 15 ft were nonviable after day 28. The G. muris cysts exposed to river water remained viable up to 28 days as determined by the production of giardiasis in mice. G. muris cysts suspended in tap water showed no signs of viability after 14 days, while cysts serving as controls (exposed to refrigerated distilled water) remained viable for up to 56 days. In the winter, Giardia cysts suspended in either lake or river water were viable for 56 to 84 days whereas cysts exposed to tap water were nonviable by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin.

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of viable Giardia cysts in vitro: determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil.

The purpose of this research was to document the formation of viable Giardia cysts in vitro. Viability staining, using fluorogenic dyes that required metabolic conversion for detection, and immunocytochemistry at the light microscopic level provided information on viability and for the identification of formed in vitro. Analysis of cysts formed in vivo and in vitro showed similar morphologic appearances by both light and electron microscopy. Cysts formed in vitro were capable of establishing infections in both mouse and gerbil models for giardiasis. Trophozoites obtained from mice experimentally infected with in vitro-formed cysts could be maintained in culture and induced a second time to form cysts in vitro. This model for the production of viable Giardia cysts in vitro should facilitate research on controlling the complete life cycle of Giardia outside an animal host.

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study.

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.

Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy.

Examination of Giardia muris cysts stained with the fluorogenic dyes, fluorescein diacetate (FDA) or propidium iodide (PI), by either Nomarski differential interference contrast (DIC), phase, or brightfield (BF) microscopy revealed a direct correlation between morphologic appearance and uptake of FDA or PI. Cysts incorporating FDA were all morphologically identical and exhibited (1) a clearly delineated cyst wall, (2) the presence of a distinct space between cyst wall and cytoplasm, and (3) flagella recognizable at one pole of the cyst. FDA-positive cysts also had a hyaline appearance of the cytoplasm (examined at multiple focal planes with DIC) that made it very difficult to detect the presence of nuclei, intracellular axonemes of flagella, or curved elements of the adhesive disc. However, PI-stained cysts possessed a distinct morphology that was clearly different from that of FDA-stained cysts. Examination of PI-stained cysts demonstrated the presence of well-defined nuclei, intracellular axonemes, and curved elements of the adhesive disc. The cytoplasm of PI-stained cysts contained a fine granular texture as opposed to the hyaline appearance of FDA-stained cysts, and no space was observed separating the cyst wall from the underlying cytoplasm in the PI cyst. This light microscopic comparison of viable FDA- and nonviable PI-stained cysts of G. muris demonstrates that 2 types of cysts can be distinguished and implies that structural differences can be used to identify these subpopulations of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method to determine Giardia cyst viability: correlation of fluorescein diacetate and propidium iodide staining with animal infectivity.

The viability of Giardia muris cysts was studied with the fluorogenic dyes fluorescein diacetate (FDA) and propidium iodide (PI). G. muris cysts were seen to fluoresce intensely green with FDA at an excitation wavelength of 450 to 490 nm. Cysts stained with PI fluoresced bright orange at an excitation wavelength of 450 to 490 nm and bright red at 545 to 546 nm. Examination of isolated G. muris cyst preparations stained with FDA-PI revealed that greater than 85% of the cysts stained green with FDA and less than 15% stained orange-red with PI. Using the mouse model for giardiasis, we inoculated FDA- or PI-stained cysts into neonatal mice. Feces were examined at days 3, 5, 8, and 11 postinoculation for the presence of cysts. Using 1,000 FDA-stained cysts as the inoculum, we detected cysts at days 5, 8, and 11 postinoculation in 19 of 19 mice, whereas a 50-fold greater dose of cysts produced infection in 27 of 27 mice at day 3 as well as at days 5, 8, and 11 postinoculation. Inoculation of mice with either 5,000 or 50,000 PI-stained G. muris cysts did not produce infection in any of the animals. Necropsy of mice infected with FDA-stained cysts showed trophozoites within the intestines. No trophozoites were detected within animals inoculated with PI-stained cysts. These results demonstrate that FDA-positive cysts are viable, as determined by infectivity, while PI-positive cysts are nonviable and incapable of producing G. muris infections in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)